Kiyoshi Kataoka

Critical levels of extracellular glutamate mediating gerbil hippocampal delayed neuronal death during hypothermia: Brain microdialysis study

When the brain temperature was lowered by 2 degrees C from normothermic temperature, a protective effect on postischemic neuronal death was exhibited and levels of extracellular glutamate were attenuated to about half of those at normothermic brain temperature in the gerbil hippocampus. Hypothermia has been reported to confer a protective effect on ischemia-induced delayed neuronal death. The present study was carried out to quantify this protective effect of hypothermia on the degree of alteration in extracellular release of glutamate during ischemia and the final histopathological outcome in the hippocampus. Extracellular glutamate levels were measured by microdialysis. In gerbils whose brain temperature was maintained at normothermia (37 degrees C), glutamate increased during ischemia and the early period of recirculation (by 15-fold), and CA1 neurons were consistently damaged. In animals whose brain temperature was maintained at 35 or 33 degrees C during ischemia, the release of glutamate was significantly attenuated to half or a quarter, respectively, at 37 degrees C. In animals whose brain temperature was maintained at 31 degrees C during ischemia, the release of glutamate was slightly lower than that at 33 degrees C. No CA1 ischemic neuronal damage was seen in 60% of gerbils at 35 degrees C and none was seen in any gerbils at 33 and 31 degrees C. In animals whose brain temperature was maintained at 39 degrees C during ischemia, the release of glutamate was slightly higher than that at 37 degrees C, and a high mortality rate of animals (75%) was observed. Our results reinforce other recent evidence suggesting that one of the mechanisms by which lowering of the brain temperature by only a few degrees during ischemia exerts a protective effect in the hippocampus, involves the reduction of ischemia-induced glutamate release.

Selective vulnerability of hippocampal CA1 neurons cannot be explained in terms of an increase in glutamate concentration during ischemia in the gerbil: Brain microdialysis study

Ischemia-induced selective neuronal injury to field CA1 is not attributable to selective glutamate release in field CA1 during ischemia. Excessive release of glutamate has been proposed to play a major role in ischemia-induced selective neuronal death in field CA1 of the hippocampus. It is well known that, following carotid arterial occlusion of 5 min duration in the gerbil, the pyramidal neurons in field CA1 show delayed neuronal death, whereas the neurons in field CA3 do not show any neuronal degeneration. In the present study, we measured the levels of released glutamate during ischemia in field CA1 and field CA3, separately, and evaluated whether there are subregional differences in the concentration of released glutamate which could be a satisfactory explanation for the selective vulnerability of hippocampal neurons to ischemia. Extracellular glutamate levels were significantly increased during ischemia in both field CA1 and field CA3. No significant differences were detected in the time-course of change in glutamate release and the levels of glutamate between field CA1 and field CA3. This result indicates that the increased glutamate levels do not play a pivotal part in the detrimental effect of glutamate during 5-min ischemia. Some differentiated post-synaptic organization may act as a crucial factor in the development of ischemia-induced selective neuronal death in the gerbil hippocampus.

Temperature dependence of hypoxia-induced calcium accumulation in gerbil hippocampal slices

Microfluorometry was used to investigate temperature dependence of hypoxia-induced intracellular calcium accumulation in gerbil hippocampal slice. When slices were superfused with hypoxic medium at 37 degrees C, 35 degrees C, 33 degrees C or 31 degrees C, latencies of acute increase of calcium accumulation, which was accompanied by a large negative shift of extracellular DC potentials, were delayed in a dose-dependent manner: mean latencies in field CA1 were 130 s, 182 s, 232 s and 277 s after hypoxia, respectively. This retardation in calcium accumulation may be involved in the mechanisms by which hypothermia diminishes ischemic injury.

Release of Excitatory Amino Acids from Cultured Hippocampal Astrocytes Induced by a Hypoxic‐Hypoglycemic Stimulation

Abstract: An excess release of excitatory amino acids (EAA) is an important factor for postischemic brain damage. In the present communication, we demonstrate that cultured hippocampal cells release EAA after hypoxic‐hypoglycemic treatment. The amounts of EAA released from astrocytes were appreciably above those released from neurons. Furthermore, the amount of aspartate released from astrocytes was comparable to that of glutamate, although the endogenous content of aspartate was one‐fifth that of glutamate. The endogenous content of aspartate in astrocytes increased even after hypoxic‐hypoglycemic treatment. These results suggests that ischemic neuronal death is due, at least in part, to the excitotoxicity of aspartate and glutamate‐derived from surrounding astrocytes.

Gerbil hippocampal extracellular glutamate and neuronal activity after transient ischemia.

In order to elucidate the role of glutamate in the pathogenesis of delayed neuronal death, we analyzed changes in extracellular levels of glutamate induced by transient ischemia in the Mongolian gerbil hippocampus by a new brain microdialysis method combined with an enzymatic cycling technique. We also studied the effect of this change in glutamate on CA1 spontaneous neuronal discharges. The level of glutamate significantly increased during the 5 min of ischemia and during the first 5 min of recirculation. However, neuronal hyperactivity anticipated as a result of the increased extracellular glutamate was not observed. Spike discharges disappeared during and shortly after 5 min of ischemia; CA1 spontaneous spike discharges reappeared about 15 min after the recirculation. The frequency and amplitude of the discharges of CA1 neurons returned to normal by 30 min of the recirculation. However, the pattern of discharges was different from that recorded before the ischemia. CA1 neurons were found dead 4 days after the ischemia. Brief exposure to toxic concentrations of glutamate may cause the delayed neuronal death.

Delayed Neuronal Death is Induced without Postischemic Hyperexcitability: Continuous Multiple-Unit Recording from Ischemic CA1 Neurons

It has been proposed that neuronal hyperexcitability during postischemic chronic stage mediates delayed neuronal death in the hippocampal CA1 region. In the present study, multiple-unit spike discharges were continuously recorded from hippocampal CA1 neurons of the awake Mongolian gerbil for 5 days after 5 min of ischemia. Before ischemia, CA1 neurons showed burst-like spike discharges (so-called complex spikes). Spike discharges disappeared 8–40 s after the onset of 5-min ischemia and reappeared 5–30 min after recirculation. The frequency of discharges gradually increased but did not return to the preischemic level. The amplitude of the spike discharges was smaller than that recorded before ischemia and the number of spikes composing the burst-like discharges diminished. CA1 neurons did not show any hyperexcitability for 5 days. However, histological examinations revealed widespread neuronal death in the CA1 region. These results indicate that the delayed neuronal death in the hippocampal CA1 region is induced without postischemic neuronal hyperexcitability.

A new enzymatic cycling technique for glutamate determination in brain microdialysates.

A quantitative analysis of glutamate in brain dialysate was made by using an enzymatic cycling technique. This method made it possible to measure the concentration of glutamate in dialysate collected at 30‐s intervals. Dialysates were collected from Mongolian gerbil hippocampus before, during, and after two 90‐s ischemic insults at an interval of 5 min. An extracellular increase in levels of glutamate was already observed in samples collected during a 30–60 s period after the onset of each ischemia, and the levels of glutamate were maximal at the end of each period of ischemia (approximately a fourfold increase). The increased levels of glutamate rapidly returned almost to preischemic levels by 30 s of recirculation. This method will provide more precise information about temporal changes in the extracellular glutamate concentration in the brain during ischemia.

Distribution of hypoxia-induced calcium accumulation in gerbil hippocampal slice

Microfluorometry was used to examine distribution of hypoxia-induced intracellular free calcium accumulations in Mongolian gerbil hippocampal slice. Acute increase of intracellular free calcium was detected 80-170 s after the beginning of hypoxia. Large calcium accumulations were seen in the stratum radiatum, stratum lacunosum and stratum oriens of CA1 region and in the inner portions of the dentate gyrus molecular layer, and moderate accumulations were seen in the other laminae of CA1 region, other portions of dentate gyrus and the CA3 region.

Visualization of hypoxia-induced glutamate release in gerbil hippocampal slice

Microfluorometry was used to investigate distribution of hypoxia-induced release of glutamate. Mongolian gerbil hippocampal slice was perfused in a medium containing glutamate dehydrogenase and NAD+. Release of glutamate into extracellular space caused an increase in fluorescence due to the formation of NADH. The hypoxia-induced release of glutamate was gradually increased throughout the slice: no significant difference was detected among CA1 region, CA3 region and the dentate gyrus.

High frequency discharges of gerbil hippocampal CA1 neurons shortly after ischemia

It has been postulated that the central neurotoxicity of glutamate participates in the pathogenesis of the ischemia-induced neuronal death and the process of the neuronal death is initiated by overexcitation or depolarization of postsynaptic neurons induced by increased extracellular glutamate during ischemia. In the present study, in order to know whether ischemic neurons show the overexcitation, we studied changes of CA1 neuronal discharges in gerbil hippocampus induced by transient forebrain ischemia (1-5 min) using an extracellular unit recording technique. CA1 neurons showed the high frequency discharges shortly after ischemic insult of 90 sec, however, these discharges did not induce neuronal death. Delayed neuronal death in the CA1 sector was observed in animals with 5-min ischemia which did not induce high frequency discharges. Neuronal depolarization with no spike discharge may persist during and shortly after 5-min ischemia and initiate the delayed neuronal death.